蚊抗藥性相關新基因NYD-Ch和NYD-Tr的初步功能研究
發(fā)布時間：2018-12-18 06:48
【摘要】：【目的】研究抗藥性相關糜蛋白酶基因(NYD-Ch)和胰蛋白酶基因(NYD-Tr)與抗藥性之間的關系。 【材料和方法】采用PCR方法,以淡色庫蚊抗性品系cDNA文庫為模板,擴增出抗性品系中高表達的NYD-Ch和NYD-Tr,經T/A克隆測序鑒定后,擴增純化質粒,經酶切后純化目的片段亞克隆到真核表達載體pIE1-3中,用PCR結合酶切鑒定,核酸測序驗證。將上述重組表達載體分別單獨轉入和共轉入蚊細胞,經篩選獲得穩(wěn)定轉染的細胞系,RT-PCR和Western blotting鑒定目的基因的穩(wěn)定表達,并用MTT和流式檢測穩(wěn)定表達的細胞對溴氰菊酯殺蟲劑的敏感性。 【結果】成功構建昆蟲細胞表達載體并建立穩(wěn)定轉染細胞系,使目的基因在其中穩(wěn)定表達。MTT結果顯示:pIE1-3/NYD-Ch單獨轉染組,細胞存活率與對照組無顯著性差異(p0.05);pIE1-3/NYD-Tr單獨轉染組,120μM溴氰菊酯濃度時,其細胞存活率較空載體組和未轉染組分別增加38.20%(p0.05)和9.66%(p0.05);pIE1-3/NYD-Ch和pIE1-3/NYD-Tr共轉染組,120μM溴氰菊酯濃度時,其細胞存活率較空載體組和未轉染組分別增加67.37%(p0.01)和38.83%(p0.001);160μM藥物濃度時,其細胞存活率較空載體組和未轉染組分別增加48.83%(p0.05)和24.26%(p0.05)。 【結論】NYD-Ch和NYD-Tr在細胞對抗溴氰菊酯的毒性作用中有一定的保護力,提示NYD-Ch和NYD-Tr與抗藥性相關。
[Abstract]:[objective] to study the relationship between drug resistance associated chymotrypsin gene (NYD-Ch) and trypsin gene (NYD-Tr) and drug resistance. [materials and methods] using cDNA library of Culex pipiens pallens resistance strain as template, the highly expressed NYD-Ch and NYD-Tr, in the resistant lines were amplified by PCR method, and the purified plasmids were amplified by T / A cloning and sequencing. The target fragment was subcloned into eukaryotic expression vector pIE1-3 after digested by enzyme, identified by PCR binding enzyme digestion and verified by nucleic acid sequencing. The recombinant expression vectors were transferred into mosquito cells separately and cotransferred into mosquito cells. Stable transfected cell lines were obtained by screening. The stable expression of the target gene was identified by RT-PCR and Western blotting. The sensitivity of stably expressed cells to deltamethrin was detected by MTT and flow cytometry. [results] insect cell expression vector was successfully constructed and stable transfection cell line was established, in which the target gene was stably expressed. MTT results showed that pIE1-3/NYD-Ch was transfected alone. There was no significant difference between the cell survival rate and the control group (p0.05). The cell survival rate of pIE1-3/NYD-Tr alone transfected with 120 渭 M deltamethrin increased by 38.20% (p0.05) and 9.66% (p0.05) respectively compared with the empty vector group and the untransfected group. The cell survival rate of pIE1-3/NYD-Ch and pIE1-3/NYD-Tr co-transfected group increased 67.37% (p0.01) and 38.83% (p0.001) respectively compared with empty vector group and untransfected group at 120 渭 M deltamethrin concentration. The cell survival rate was increased by 48.83% (p0.05) and 24.26% (p0.05) respectively at 160 渭 M concentration compared with empty vector group and untransfected group. [conclusion] NYD-Ch and NYD-Tr have protective effect on the cytotoxicity of deltamethrin, suggesting that NYD-Ch and NYD-Tr are related to drug resistance.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R384.1
本文編號：2385514
[Abstract]:[objective] to study the relationship between drug resistance associated chymotrypsin gene (NYD-Ch) and trypsin gene (NYD-Tr) and drug resistance. [materials and methods] using cDNA library of Culex pipiens pallens resistance strain as template, the highly expressed NYD-Ch and NYD-Tr, in the resistant lines were amplified by PCR method, and the purified plasmids were amplified by T / A cloning and sequencing. The target fragment was subcloned into eukaryotic expression vector pIE1-3 after digested by enzyme, identified by PCR binding enzyme digestion and verified by nucleic acid sequencing. The recombinant expression vectors were transferred into mosquito cells separately and cotransferred into mosquito cells. Stable transfected cell lines were obtained by screening. The stable expression of the target gene was identified by RT-PCR and Western blotting. The sensitivity of stably expressed cells to deltamethrin was detected by MTT and flow cytometry. [results] insect cell expression vector was successfully constructed and stable transfection cell line was established, in which the target gene was stably expressed. MTT results showed that pIE1-3/NYD-Ch was transfected alone. There was no significant difference between the cell survival rate and the control group (p0.05). The cell survival rate of pIE1-3/NYD-Tr alone transfected with 120 渭 M deltamethrin increased by 38.20% (p0.05) and 9.66% (p0.05) respectively compared with the empty vector group and the untransfected group. The cell survival rate of pIE1-3/NYD-Ch and pIE1-3/NYD-Tr co-transfected group increased 67.37% (p0.01) and 38.83% (p0.001) respectively compared with empty vector group and untransfected group at 120 渭 M deltamethrin concentration. The cell survival rate was increased by 48.83% (p0.05) and 24.26% (p0.05) respectively at 160 渭 M concentration compared with empty vector group and untransfected group. [conclusion] NYD-Ch and NYD-Tr have protective effect on the cytotoxicity of deltamethrin, suggesting that NYD-Ch and NYD-Tr are related to drug resistance.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R384.1
【引證文獻】
相關博士學位論文 前1條
1 胡小邦;視蛋白基因的克隆及其與抗藥性/耐藥性關系的研究[D];南京醫(yī)科大學;2007年
相關碩士學位論文 前1條
1 段曉雷;PR-XP1基因的克隆及在C6/36耐藥細胞株中抗性的初步研究[D];西北農林科技大學;2011年
，本文編號：2385514
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